Case 3 -
The Children's Hospital
University of Colorado Denver
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A previously healthy 2-year old female presented with a 1-month history of progressive bilateral lower
extremity weakness and a neurogenic bladder. Imaging revealed an epidural tumor, which was biopsied and
debulked to relieve the symptoms of spinal cord compression. Metastatic work-up was negative. She
underwent chemotherapy and radiotherapy with good response and is currently in disease remission 4 years
Case 3 - Slide 1
Case 3 - Figure 1
Touch preparation performed on tumor biopsy tissue, showing discohesive population of relatively uniform, small to medium-sized cells with high nuclear/cytoplasmic ratio and apoptotic activity.
Case 3 - Figure 2
Low-power histomorphologic features of tumor, showing a dense, small blue cell population with patchy necrosis, infiltrating fibrocollagenous tissue.
Case 3 - Figure 3
Intermediate-power histomorphologic features of tumor, showing a relatively uniform cell population with clear cytoplasm.
Case 3 - Figure 4
High-power histomorphologic features of tumor, showing cells with fine chromatin, inconspicuous nuclei, amphophilic to clear cytoplasm and scattered apoptotic activity.
Case 3 - Figure 5
CD99 immunohistochemical staining of tumor, showing uniform membranous staining of tumor cells in well-preserved areas.
Case 3 - Figure 6
Higher-power view of tumor CD99 immunostaining.
Case 3 - Figure 7
Ultrastructural features of tumor, showing cells with high nuclear-cytoplasmic ratio, and organelle-poor cytoplasm containing abundant glycogen in the polyparticulate form.
The biopsy consisted of multiple pieces of soft tissue. Touch preparation revealed a discohesive
population of relatively uniform small to medium-sized cells with high nuclear/cytoplasmic ratio and
apoptotic activity. Histologic examination showed a dense population of malignant small blue cells with
clear to amphophilic cytoplasm, punctate chromatin, small nucleoli, and scattered apoptotic and mitotic
activity. Immunophenotyping revealed the tumor cells to be strongly and diffusely positive for CD99 in a
crisp membranous pattern, and immunongative for pancytokeratin, synaptophysin, neuron-specific enolase,
desmin, myogenin, CD45 and TdT. Electron microscopic examination showed poorly differentiated cells
containing abundant cytoplasmic glycogen in the polyparticulate form. Fluorescence in-situ hybridization
studies revealed rearrangement of the EWSR locus, and molecular diagnostic studies demonstrated the
presence of the EWS/Fli1 oncogenic fusion transcript. Together, these findings unequivocally established
the diagnosis of Ewing Sarcoma/ Peripheral Primitive Neuroectodermal Tumor (PNET).
Ewing Sarcoma is an enigmatic malignancy. First described by James Ewing in 1921 as an
endothelioma of bone, the histogenesis of this aggressive, poorly differentiated tumor remains uncertain
to this day. Ewing Sarcoma is a tumor of bone and soft tissue, and less commonly viscera. The current
definition encompasses the entities of Ewing Sarcoma, Peripheral Primitive Neuroectodermal Tumor,
Peripheral Neuroepithelioma and Askin Tumor, all of which share the same EWS/Ets oncogenic fusions and
similar biologic behavior. Ewing Sarcoma represents the second most common bone and soft tissue
malignancy in adolescents and young adults, peaking in incidence at about 5 per million in this age
group. It is slightly more common in males, more common in Caucasians and rarely arises in individuals
of African ancestry. As with other bone and soft tissue malignancies, Ewing Sarcoma presents with pain
and/or a mass, and a destructive/infiltrative lesion on imaging. The most common bony sites are the long
bones of the extremities, pelvis, chest wall and spine. Lesions of long bones typically involve the
Pathologic examination is key to diagnosis. Well-preserved biopsy specimens show
characteristic histomorphology and ultrastructure, as exemplified in the above case, including relatively
uniform, malignant, small to medium-sized cells with little amphophilic to clear cytoplasm, punctate
chromatin, inconspicuous nuclei and abundant cytoplasmic glycogen in the polyparticulate form.
Immunophenotyping helps to support the diagnosis. Well-preserved specimens are positive for CD99 in a
diffuse membranous pattern. A number of other tumors may show varying degrees of CD99 immunopositivity,
but in an experienced laboratory, diffuse CD99 positivity in a membranous pattern is highly suggestive of
the diagnosis of Ewing Sarcoma in the right morphologic context. The tumor may also be variably positive
for cytokeratin, synaptophysin and neuron-specific enolase. In pediatric patients, the main differential
diagnosis includes leukemia/lymphoma, poorly differentiated rhabdomyosarcoma, and, depending on site,
undifferentiated neuroblastoma; these diagnoses are excluded by CD45 and TdT negativity, myogenin and
desmin negativity, and CD99 positivity, respectively. Occasional Ewing Sarcomas may show divergent
skeletal muscle differentiation, and one must rely on cytogenetic/molecular studies (presence of EWS/Ets
fusion) to arrive at the correct diagnosis. For bone lesions, absence of malignant osteoid further
excludes small cell osteosarcoma; similarly, absence of a cartilaginous component helps exclude
mesenchymal chondrosarcoma. On small biopsies, additional diagnostic considerations may include poorly
differentiated synovial sarcoma (SS) and desmoplastic small round cell tumor (DSRCT); these can be
differentiated by immunophenotyping (desmin positivity and CD99 negativity in DSRCT) and
cytogenetic/molecular studies (X;18 translocation in SS and EWS/WT1 oncogenic fusion in DSRCT).
Ultimately, if cytogenetic and/or molecular studies are available, the diagnosis of Ewing Sarcoma can be
confirmed, and alternative diagnoses excluded, by the demonstration of an EWS/Ets oncogenic fusion. When
available, ultrastructural studies can also be very helpful in navigating the differential diagnosis of
Ewing Sarcoma. Occasionally one encounters Ewing-like tumors that lack the characteristic
immunophenotype and (known) EWS/Ets fusions; these are diagnosed as unclassified/undifferentiated small
round cell malignancies/sarcomas. This scenario underscores the importance of allocating fresh biopsy
tissue for additional (cytogenetic, molecular and ultrastructural), studies whenever possible, as this
may result in the identification of novel diagnostic features in such cases. In the not-so-distant
future, such tumors may also be further subclassified by gene expression profiling.
The two most important prognostic indicators in the staging of Ewing Sarcoma are tumor
size (greater or less than 8 cm) and presence or absence of metastatic disease. Additional prognostic
information is conferred by fusion type: for tumors harboring the EWS/Fli1 fusion, the type 1 variant
carries a better prognosis. More recently, additional molecular indicators of (poor) prognosis have been
identified, including p53 mutation and p16/p14ARF deletion. Further, prognostically meaningful tumor "signatures" that
predict disease outcome are emerging from global gene expression profiling studies. From the standpoint
of treatment, the presence of absence of overt metastasis is the most important determinant, although all
patients are presumed to harbor micrometastatic disease. Multi-agent chemotherapy has raised survival
for patients without evidence of overt metastasis at the time of presentation from 10% to 50-60% at 5
years. However, survival for the 25% of patients with overt metastatic disease at presentation is less
than 25% at 5 years; recurrent disease carries a similarly grim prognosis. Treatment of patients without
overt metastatic disease consists of initial chemotherapy (for cytoreduction and eradication of any
micrometastatic disease), followed by surgical resection, and then consolidation chemotherapy (to reduce
the likelihood of recurrence). Treatment options for metastatic and recurrent disease include
chemotherapy, surgery, radiation, biologically targeted therapy and bone marrow transplantation, but
little progress has been made in improving survival in this group. Hence, there is a tremendous
potential role for new therapies.
Biologically, Ewing Sarcoma is the canonical example of a malignancy driven by a fusion
oncogene. Ewing Sarcomas harbor fusions between the amino terminus of the EWS gene on chromosome 22 and
the carboxy terminus of one of five Ets family genes. The Ets gene is Fli1 in 85% of cases, Erg in 10%
of cases, and Etv1, Etv4 or FEV in the remaining 5% of cases. EWS is a member of the TET family of
proteins, which are ubiquitously expressed in all cells, and appear to be involved in transcription
and/or RNA processing. Fli1 and the other Ets genes are tissue-specific transcription factors. In-frame
fusion of EWS to an Ets factor in Ewing Sarcoma yields a highly expressed, non-physiologic, potent
transcription factor, which dysregulates gene expression in the cell.
All experimental evidence points to EWS/Ets fusions being the key oncogenes in Ewing
Sarcoma. Namely, EWS/Ets fusions are both necessary and sufficient, in the appropriate cellular context,
to drive oncogenesis. Thus, the key to understanding Ewing Sarcoma pathogenesis centers on the biology
of these fusions. A shortcoming of much early research on Ewing Sarcoma was the lack of experimental
systems that closely model the disease. Recently, a number of advances have been made on this front.
First, RNA interference technology has permitted the identification of bona
fide cellular pathways mediating EWS/Ets oncogenicity, opening the way to the identification of
new "druggable targets" for therapy. Second, two independent groups have shown that expression of
EWS/Fli1 in bone marrow-derived mesenchymal stem cells is sufficient to induce Ewing Sarcoma-like tumors
in a mouse xenograft model. In a related study, another group has shown that loss of EWS/Fli1 expression
in Ewing Sarcoma results in change to a mesenchymal progenitor-type cell with multipotential
differentiation capacity. Although not proof, these findings offer strong evidence in support of a
mesenchymal stem cell origin for Ewing Sarcoma. This, in turn, has given rise to better experimental
models, and finally opened the door to the possibility of an animal genetic model for the disease. The
hope, of course, is that better understanding of Ewing Sarcoma pathogenesis through improved experimental
systems will yield more effective therapies. To this end, identification of insulin-like growth factor
signaling as essential for Ewing Sarcoma-genesis, has already led to biologically based therapies
currently in Phase II clinical trials, with more likely to follow. The "holy grail" for a biologically
targeted therapy remains blockade of the oncogenic EWS/Ets fusions themselves, a goal toward which,
unfortunately, little progress has been made to date.
- Ewing Sarcoma is an aggressive malignancy affecting adolescents and young adults
- Diagnosis is suggested by characteristic histomorphology, ultrastructure and immunophenotype, and confirmed by cytogenetic and/or molecular analysis
- Metastatic and recurrent Ewing Sarcoma remains a disease with poor prognosis
- EWS/Ets oncogenic fusions drive Ewing Sarcoma-genesis
- Recent evidence supports a mesenchymal stem cell origin for Ewing Sarcoma
- New model experimental systems offer hope for the discovery of more effective therapies
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